1. Field of the Invention
The invention relates generally to the field of nanotechnology. More particularly, the invention relates to nanostructures, methods of preparing and interfacing nanostructures with biological systems, and devices for implementing such interfaces.
2. Discussion of the Related Art
Efficient manipulation of biological systems at the subcellular and, ultimately, molecular scale is a critical need to enable mankind to further its knowledge of cellular processes and to enhance our ability to modify and control cellular function. Much of the data on cellular processes were obtained by cell culture and study, where stimuli are applied to cells (chemical, electrical, electrochemical, thermal, mechanical, etc) and the cellular responses to stimuli are directly observed using a variety of techniques (microscopy, fluorimetry, electrochemistry, etc).
A variety of techniques exist for material delivery to live cells, particularly plasmid delivery. These methods are discussed and compared in the literature. A variety of techniques also exist for single element intracellular probing or material delivery, including traditional microinjection, pulled capillary or pulled fiber optic probes, and single carbon tube bundle electrophysiology. Parallel interfacing techniques also exist based largely upon culturing cells upon planar arrays of electrodes.
Often, research is conducted by observing a single specimen, or a single cell, at one time, applying a stimulus and observing a response. As the response of a single specimen may be dramatically different from the norm, many individual trials must be conducted to obtain information of normal response. Methodologies that would provide parallelism to such studies can significantly streamline effort and cost, and can dramatically improve the rate of knowledge gain.
Among the many methods that may be used to deliver DNA to a targeted cell, perhaps the most straightforward, but most effective, is microinjection—the direct administration of naked DNA to the intracellular domain of a targeted cell [27]. Microinjection forcibly bypasses the physical barriers that a cell relies upon to protect itself from foreign DNA, and in so doing, enables high probability for transformation success. Used routinely for eukaryotic transformation including both mammalian and plant cells, recent success with ultrafine micropipettes (<100 nm tip diameters) has been reported on prokaryotes and even targeted organelles [11]. The task of microinjection, however, is time consuming and arduous as the precision of the method requires the targeting of an individual cell and typically its nuclear envelope using fragile micropipettes and precise micromanipulators.
Parallel techniques for DNA delivery have naturally developed to streamline the process of engineering the cell. While the ultimate goal of these techniques is typically the delivery, stable insertion, and expression of exogenous DNA into a target organism, most methods typically only address one or two components of a complex, multistep process. As reviewed by Luo and Saltzman [27], once a cell is targeted, successful delivery requires that DNA breach the plasma membrane, run (or at least survive) the gauntlet of intracellular endosomal/lysosomal pathways, and (with some exceptions) pass yet another barrier, the nuclear envelope. Microinjection successfully addresses all of these components, including targeting of individual cells and subsequent breaching of the plasma and nuclear membrane, thereby eliminating the chance for cytosolic degradation of delivered vectors via endosomal/lysosomal pathways. In contrast to microinjection, very few methods target individual or select groups of cells, but instead approach DNA delivery as a bulk process. Electroporation, sonoporation, DNA complexing and/or precipitation, and microprojectile bombardment are all designed as bulk processes where a vector or multiplexed vectors are delivered to cells with little potential for targeting individual groups. Similarly, while viral mediated methods are highly efficient, and may be used to target specific cells or cell types, they provide no ability to target individual groups of cells within a cellular matrix. Whisker or fiber-mediated methods have also evolved that typically employ small DNA-coated fibers (carbon) or whiskers (silicon carbide) that are physically vortexed in suspensions of cells, causing localized abrasions and ultimately some unpatterned delivery to cells in the suspension. These latter methods have been typically targeted at plant cells that feature a rigid cell wall as an additional barrier to intracellular delivery.
In recent years, a large funding base directed towards gene therapy and pharmaceutical exploration combined with advances in microscale fabrication has focused a sector of the research community on developing devices and improved methods for delivering large molecules (drugs and genetic vectors) into intact cells and tissue. To date, many of these efforts have focused on the development of consumer-oriented drug-delivery platforms by utilizing the miniaturization afforded by microscale fabrication. Examples include arrays of microscale ‘painless’ needles for transdermal drug delivery [12] or microfabricated insulin vesicles that conceivably could interface with the bloodstream and provide insulin infusion when required [31].
While microscale technologies are and will continue to produce dramatic advances in biological and biomedical applications, the nanoscale, not the microscale, is the proper size regime for a direct interface to the molecular mechanisms of cells. McAllister's painless needle, an array of needles produced by reactive ion etching of silicon, features 100 micron long needles with tip diameters of microns. While these needles can effectively penetrate the stratum corneum (the dead outer layer of skin) and deliver material to the interstitial fluid and capillary beds of the epidermis, they are too large for direct material delivery into cells.
The research community has developed a variety of techniques for delivering large molecules into cells, and continues to actively seek new methods for both in-vitro (for laboratory research) and in-vivo (for clinical application) material delivery. Traditional methods rely on either natural biological mechanisms or coarse, mechanically-based means of disrupting the cellular envelope to enable passage of extracellular material. The biological methods, employing bacterial or viral infection as the transformation mechanism (i.e transfection), are only narrowly applicable, working selectively on organisms that act as hosts to the infective vector. The mechanical methods—including electro/sono/optoporation, silicon carbide whisker mediated delivery, microprojectile bombardment, and microinjection—operate on the principle of cellular envelope disruption or physically penetrating the membrane barrier. Except for microinjection, these methods are employed on bulk volumes of cells. While powerful (and sometimes highly efficient), these methods lack the highly parallel and site-specific delivery of molecular material to cells that is the key requirement for bringing powerful combinatorial techniques to the molecular manipulations of cells. As a result, research that investigates cellular response to a variety of different macromolecular reagents requires manipulating many different samples, isolation and culture of these individual samples, and careful practice to ensure samples are treated similarly (to enable true comparison of results between samples). Experiments evaluating a large array of different macromolecules to be delivered (such as gene delivery for the investigation of the functions of gene groups with single base polymorphisms) can quickly become overwhelming in terms of effort and cost. The tools the inventors propose here transform these serial operations into parallel—actually massively parallel—operations. Similar tools for combinatorial chemistry revolutionized therapeutic drug discovery and are making significant impacts in numerous other fields.
Recently, researchers at the Whitehead Institute have described a powerful new combinatorial-based approach that provides for spatially resolved cellular transformation of cultured mammalian cells [33]. In brief, cells are cultured directly onto microscope slides that have been prepared with isolated spotted regions of assorted plasmid DNAs immobilized within a gelatin matrix. Using a lipid transfection agent, discrete groups of cells grown upon these isolated regions are able to uptake, and express, the exogenous plasmid. While limited to a subset of mammalian cells that may be directly cultured and that directly uptake naked DNA, this combinatorial method has an enormous potential for rapidly screening large sets of cDNAs or DNA constructs by directly observing phenotypic changes of discrete regions of identically handled cells. Expanding upon the Whitehead study to provide a method for evaluating gene function using a larger variety of cell types (and possibly tissue) would dramatically strengthen the power of these combinatorial methods.
Microinjection is perhaps the most reliable tool for material delivery at the single cell level. As microinjection is simply using a microscale needle to penetrate the cellular barrier and deliver material, this technique is perhaps the most universally applicable method for a variety of different cell types. While implementation is technically challenging (requiring micromanipulation of pulled capillaries and cells under an optical microscope) and throughput is very low, delivery efficiency is typically very high on a cell to cell basis. A somewhat analogous method is used in bulk solutions, ‘silicon carbide whisker mediated transformation’. Plasmid/cell suspensions are formulated with small amounts of silicon carbide whiskers (microscale needlelike fibers) that, when agitated, promote material delivery by creating lesions in the walls and membranes of cells.